Leptin active peptide having cd loop and E helix region mutations, coding gene thereof, and application thereof

ABSTRACT

A leptin active peptide having CD-loop and helix E region mutations, a coding gene thereof, and an application thereof are provided in the present invention. An amino acid sequence of the leptin active peptide having CD-loop and helix E region mutations is shown in SEQ ID NO.1.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Patent ApplicationNo. PCT/CN2015/088554 with a filing date of Aug. 31, 2015, designatingthe United States, now pending, and further claims priority to ChinesePatent Application No. 20151022093.8 with a filing date of May 6, 2015.The content of the aforementioned applications, including anyintervening amendments thereto, are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention belongs to the fields of biochemistry andmolecular biology, and particularly relates to a leptin active peptidehaving CD-loop and helix E region mutations, a coding gene thereof, andan application thereof.

BACKGROUND OF THE INVENTION

Obesity is one of the worst enemies of human health. Obesity is anindependent disease as well as a pathogenic cause of type II diabetes,cardiovascular diseases, hypertension, cerebral apoplexy and multiplecancers, is listed as one of top ten threats causing disease burdens byWorld Health Organization, and is a global issue that cannot be ignored.It is reported that about 18 million people die from cardiovascular andcerebrovascular diseases caused by diabetes and hypertension every yearin the world, while the obesity is a key pathogenic factor of thediabetes and hypertension. The obesity seriously endangers human healthand causes huge losses to the society. It is reported in 2003 that,expenditure for chronic diseases such as the obesity, the diabetes andthe like reaches RMB 1200 billion in China and accounts for 10.3% ofGDP. An increasing rate of the expenditure is higher than an increasingrate of Gross National Product (GNP). At present, domestic weight lossmarket needs are very wide. Consumption amount of weight loss productsreaches RMB 60 billion in 2010, while losses caused by diseases, such asthe diabetes, cardiovascular diseases and the like, directly orindirectly related to the obesity are beyond calculation. At present,although drugs for treating the obesity and the diabetes, i.e.,fenofibrate and rosiglitazone, can achieve effects of reducing blood fatand blood sugar, serious side effects, such as insulin resistance,“mutagenesis, carcinogenesis and teratogenesis” effects and the like,may be caused to a human body when the drugs are taken for a long time.Therefore, safe, efficient and cheap novel drugs are urgently needed inclinical treatment of the obesity and the diabetes.

Leptin is a hormone substance secreted by adipose tissue. Six leptinreceptors participate in fat metabolism and appetite regulation of theorganism. At the beginning of this century, the leptin is discovered andsuccessfully tested on a mouse model suffering from the obesity. A stirin scientific circles is created. It is forecast that biotechnologyfinds a novel weight loss drug for hundreds of millions of obesepatients around the world. In 1999, through study, scientists have foundout that polypeptide fragments of 116th-130th amino acid tissues of ahuman-derived leptin protein have obvious lipid degradation activities.Later, American scientists first carried out the first leptin proteinexperiment on human being. However, in 73 tested obese patients, most ofthe patients do not have an obvious weight loss effect, and only 2patients lose weight by 35 pounds within 24 weeks, which indicates thatan ideal weight loss effect is difficult to be achieved by using a wildtype human-derived leptin protein.

SUMMARY OF THE INVENTION

A first objective of the present invention is to provide a leptin activepeptide having CD-loop and helix E region mutations with excellentadipocyte-degrading activity.

An amino acid sequence of the leptin active peptide having CD-loop andhelix E region mutations in the present invention is shown in SEQ IDNO.1.

A second objective of the present invention is to provide a coding geneof the leptin active peptide having CD-loop and helix E regionmutations.

A third objective of the present invention is to provide an applicationof the leptin active peptide having CD-loop and helix E region mutationsin preparation of adipocyte-degrading drugs, weight loss drugs orhypoglycemic drugs.

In the present invention, an amino acid sequence of human-derived leptinis modified for increasing lipid degradation efficiency. The leptinactive peptide having CD-loop and helix E region mutations issynthesized by utilizing chemical methods respectively, and an influenceof a protein space structure on an activity of the leptin is reduced asmuch as possible, thereby enhancing targeting and degrading effects onadipocytes. Practical results indicate that the adipocyte-degradingactivity of the leptin active peptide having CD-loop and helix E regionmutations in the invention is obviously better than that of positivecontrol-human-derived leptin protein (H-LEP) and a commerciallyavailable drug rosiglitazone (Ros,); the lipid-lowering activity of theleptin active peptide has obvious concentration dependence anddependence in a cell differentiation period; cell lysis effects of theleptin active peptide at three stages, i.e., a preadipocytes stage, adifferentiation stage (D0-D2) and a mature adipocyte stage (D9) areobviously better than those of two positive controls (H-LEP orrosiglitazone), particularly the lipid-lowering activity of the leptinactive peptide having CD-loop and helix E region mutations has anoptimal effect at the D0-D2 stage when the concentration is 10⁻⁶M,thereby providing a candidate target for developing novel, efficient andcheap weight loss and blood-sugar-reducing drugs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a design of a leptin active peptide having CD-loop and helix Fregion mutations; A: a three-dimensional structural diagram simulatedand constructed by taking a human-derived leptin sequence as a template;B: amino acid sequence alignment and function division of No. 3 exon ofthe human-derived leptin (upper) and the leptin active peptide havingCD-loop and helix E region mutations (lower); *: mutation site;

FIG. 2 shows activity analysis of a leptin active peptide having CD-loopand helix E region mutations; Lip-lowering activity analysis of 3T3-L1preadipocytes (MTT detection); PEP-E: the leptin active peptide havingCD-loop and helix E region mutations, as well as 10⁻⁶, 10⁻⁹ and 10⁻¹¹after the PEP-E respectively refer to concentrations, such as 10⁻⁶M,10⁻⁹M and 10⁻¹¹ M, of the leptin active peptide having CD-loop and helixE region mutations; H-LEP: human-derived leptin protein (positivecontrol), and 10⁻⁹ refers to 10⁻⁹M; Ros: rosiglitazone (positive control2); Control: negative control. Preadipocytes: induced preadipocytesstage; D0-D2: 0-2nd day of induced differentiation (an early stage ofdifferentiation); D9: the 9th day of induced differentiation (a matureadipocyte stage). Statistical analysis: a, positive control(H-LEP/Ros)vs. negative control (Control), adipocyte lysis activity ofthe positive control is obviously better than that of the negativecontrol, P<0.05; b, PEP-E vs. H-LEP, adipocyte lysis activity of thePEP-E is obviously better than that of the H-LEP, P<0.05; c, PEP-E vs.Ros, and adipocyte lysis activity of the PEP-E is obviously better thanthat of the Ros. P<0.05; and*, P<0.01.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Embodiments below are further descriptions of the present invention,rather than limitations to the present invention.

Embodiment 1

1. Material and Method

1.1. Design and Synthesis of Leptin Active Peptide Having CD-Loop andHelix E Region Mutations

A leptin active peptide having CD-loop and helix E region mutations isdesigned in a functional region thereof according tohydrophilic/hydrophobic property change conditions of mutation sites bytaking, an amino acid sequence of human-derived leptin as a template,and the amino acid sequence is SCHLHWAPGLETLDSGVQEASGY (specificallyshown as SEQ ID NO.1). The leptin active peptide having CD-loop andhelix E region mutations is synthesized by Shanghai Sangon Biotech Co.,Ltd. Through HPLC detection, a concentration of the chemosyntheticleptin active peptide having CD-loop and helix E region mutations isgreater than 23%, and a requirement for subsequent activity analysis ismet. The accuracy of the synthesized leptin active peptide havingCD-loop and helix E region mutations is detected by mass spectrometry,and the amino acid sequence of the leptin active peptide is confirmed asSCHLHWAPGLETLDSGVQEASGY (specifically shown as SEQ ID NO.1).

1.2 In-Vitro Lipid-Lowering Activity Analysis of the Leptin ActivePeptide

Lipid degradation activity of the synthesized leptin active peptidehaving CD-loop and helix E region mutations is analyzed on a cellularlevel by taking a 3T3-L1 preadipocyte line as a model. Specificoperations are as follows:

1.2.1 Experimental Design

(1) Grouping:

a: preadipocytes;

b: an early differentiation stage D0-D2 (0-2nd day of induceddifferentiation); and

c: a mature adipocyte stage D9 (the 9th day of induced differentiation);

(2) Leptin Peptide and Control

a. leptin active peptide having CD-loop and helix E region mutations(PEP-E): diluted to 10⁻³M with purified water and preserved at −20degree C. for later use. After an experiment is started, workingconcentrations have 3 concentration gradients, that is, 10⁻⁶M, 10⁻⁹M and10⁻¹¹M;

b. positive control 1: H-LEP recombinant human-derived leptin protein(10221-HNAE, Beijing Sino Biological Inc.), diluted to 10⁻⁶M withpurified water and preserved at −20 degree C. for later use. After anexperiment is started, the working concentration is 10⁻⁹M.

c. positive control 2: Ros rosiglitazone, R2408, Sigma), diluted to astock solution with a concentration of 2518 μM with DMSO and preservedat −20 degree C. for later use. After an experiment is started, thestock solution is diluted to a working solution of 0.5 μM with cellculture fluid.

d. negative control (Control): equivalent quantity of PBS

(3) Cell Differentiation Induced Liquid

a. cell differentiation induced liquid I: a complete medium containing0.5 μM IBMX(3-Isobutyl-1-methylxanthine, 15879, Sigma), 1 μMdexamethasone (dexamethasone, D4902, Sigma) and 167 nM insulin (insulin,15500, Sigma.);

b. cell differentiation induced liquid II: a complete medium containing167 nM insulin (insulin, 15500, Sigma).

1.2.2 3T3-L Preadipocyte Culture

(1) a complete culture solution: DMEM culture medium of 10% FBS (fetalcalf serum)-DMEM 45 ml+FBS 5 ml+double-antibody 0.5-0.8 ml (DMEM,Hyclone SH30243.01B500 ml high glucose, containing L-glutamine, sodiumpyruvate, Fetal Bovine Serum, Invitrogen 10091-148,Penicillin-Streptomycin, Invitrogen 15140-122 100.times.).

(2) passage: sucking old liquid, washing twice withcalcium-magnesium-free PBS, adding 0.5 ml/T25 0.25% pancreatin,digesting for about 2 min (when most of cells float), and stoppingdigestion with complete culture solution; directly inoculating in anovel 25 cm² culture bottle (T25) according to a ratio of 1:3 withoutcentrifuging; and changing the liquid after 3-4 hours, and performingpassage in time while growing by about 70%.

1.2.3 Induced Differentiation of 3T3-L1 Preadipocytes into MatureAdipocytes

(1) inoculating 3T3-L1 preadipocytes in coming cell bind 96-well plates(totally inoculated with 3 plates, respectively plate a, plate b andplate c), wherein the inoculation density is 4500 cells/well; andmarking, and standing and culturing at 37 degree C. in a 5% CO₂incubator. Each sample of each plate is operated repeatedly for 3 times,and the whole experiment is repeated for 3 times;

(2) continuously culturing for 48 hours after the cells are merged to100% (enabling the cells to exit from a growth cycle, and startinginduced differentiation.); and

(3) after the cells exit from the growth cycle,

a. preadipocytes:

i, taking out the plate a, respectively adding different amounts ofleptin active peptide having CD-loop and helix E region mutations(PEP-E) to enable the working concentrations to be respectively 3concentration gradients, that is, 10⁻⁶M, 10⁻⁹M and 10⁻¹M, setting 2positive controls, a negative control (PBS) and a blank control (3repeated holes are set for each sample), and continuously standing andculturing at 37 degree C. in the 5% CO₂ incubator.

ii, when culturing to 45 hours (that is, 3 hours before the end of drugtreatment), taking out the plate a, and measuring MTT (preadipocytes).Specific operations: adding 20 μL of 5 mg/mL of MTT solution into eachwell, continuously standing and culturing at 37 degree C. in the 5% CO₂incubator for 3 hours, sucking and discarding the culture solution inthe wells, adding 150 μL of DMSO into each well, horizontally shakingfor 10 min, detecting a light absorption value at 492 nm by a microplatereader, and drawing a growth curve.

b, the early differentiation stage (D0-D2):

i, taking out the plate b; adding the differentiation induced liquid I(D0); respectively adding the leptin active peptide having CD-loop andhelix E region mutations (PEP-E,); setting 2 positive controls, anegative control (PBS) and a blank control (3 repeated holes are set foreach sample), and continuously standing and culturing at 37 degree C. inthe 5% CO₂ incubator.

ii, when culturing to 45 hours (that is, 3 hours before the end of drugtreatment), taking out the plate b, and measuring MTT (at the earlydifferentiation stage, D0-D2). Specific operations: adding 20 μL of 5mg/mL of MTT solution into each well; continuously standing andculturing at 37 degree C. in the 5% CO₂ incubator for 3 hours, suckingand discarding the culture solution in the wells; adding 150 μL of DMSOinto each well, horizontally shaking for 10 min, detecting a lightabsorption value at 492 nm by a microplate reader, and drawing a growthcurve;

c. the mature adipocyte stage (D9):

i, taking out the plate c, adding the differentiation induced liquid I(D0) into each well; and continuously standing and culturing at 37degree C. in the 5% CO₂ incubator for 48 hours;

ii, after treating with the differentiation induced liquid I (D2) for 48hours, taking out the plate c, adding the differentiation induced liquidII into each well, and continuously standing and culturing at 37 degreeC. in the 5% CO₂ incubator;

iii, after treating with the differentiation induced liquid II (D4) for48 hours, taking out the plate c and replacing with a normal completemedium, and continuously standing and culturing at 37 degree C. in the5% CO₂ incubator;

vi, culturing for 48 hours (D6) after replacing with the normal completemedium, taking out the plate c to replace the liquid, and c standing andculturing at 37 degree C. in the 5% CO₂ incubator for 24 hours;

v, culturing to D7 (the 7^(th) day,); taking out the plate c to replacethe liquid (a complete medium.); respectively adding the leptin activepeptide having CD-loop and helix E region mutations (PEP-E), setting 2positive controls, a negative control (PBS) and a blank control (3repeated holes are set for each sample), and continuously standing andculturing at 37 degree C. in the 5% CO2 incubator for 48 hours; and

iv, when treating with drugs for 45 hours (that is, 3 hours before theend of drug treatment), taking out the plate c, and measuring MTT(mature adipocyte stage, D9), Specific operations: adding 20 μL of 5mg/mL of MTT solution into each well, continuously standing andculturing at 37 degree C. in the 5% CO₂ incubator for 3 hours, suckingand discarding the culture solution in the wells, adding 150 μL of DMSOinto each well, horizontally shaking for 10 min, detecting a lightabsorption value at 492 nm by a microplate reader, and drawing a growthcurve.

1.2.4 Statistical Analysis

The MTT detection data is subjected to one-way analysis of variance(ANOVA) by using SPSS17.0 software.

2. Results

2.1 Design and Synthesis of Leptin Active Peptide Having CD-Loop andHelix E Region Mutations

The leptin active peptide having CD-loop and helix E region mutations isdesigned and has 3 amino acid mutation sites compared with human-derivedleptin. A peptide sequence is as follows: SCHLHWAPGLETLDSGVQEASGY (shownin SEQ ID NO. 1, molecular weight: 2457.66, see FIG. 1 for details).Through HPLC detection, a concentration of the chemosynthetic leptinactive peptide having CD-loop and helix E region mutations is greaterthan 95.23%, and a requirement for subsequent activity analysis is met.

2.2 MTT Lipid-Lowering Activity Analysis

Cell activity analysis results show that the adipocyte-degradingactivity of the synthesized leptin active peptide having CD-loop andhelix E region mutations is obviously better than that of positivecontrol-human-derived leptin protein (H-LEP) and a commerciallyavailable drug rosiglitazone (Ros,) at 3 cell differentiation stages(that is, the preadipocytes stage, the early differentiation stage andthe mature adipocyte stage), and the lipid-lowering activity of theleptin active peptide having CD-loop and helix E region mutations has anoptimal effect at the D0-D2 stage when the concentration is 10⁻⁶M. (FIG.2).

SEQ ID NO. 1

Sequence table <110> SUN YAT-SEN UNIVERSITY <120>LEPTIN ACTIVE PEPTIDE HAVING CD LOOP AND E HELIX REGIONMUTATIONS, CODING GENE THEREOF, AND APPLICATION THEREOF <130>D-ZSDX-00201-NUS <160> 1  <170> PatentIn version 15 <210> 1 <211> 23<212> PRT <213> Artificial Sequence <220> <223>An amino acid sequence of the leptin active peptide havingCD-loop and helix E region mutations <400> 1Ser Cys His Leu His Trp Ala Pro Gly Leu Glu Thr Leu Asp Ser 1               5                   10                  15Gly Val Gln Glu Ala Ser Gly Tyr

We claim:
 1. A leptin active peptide having CD-loop and helix E regionmutations, wherein said peptide comprises the amino acid sequence of SEQID NO: 1.